ABSTRACT
OBJECTIVES: Our aim was to investigate pathophysiology of pseudoaneurysm of the thoracic aorta, an acute or chronic pathology, respectively, secondary to blunt thoracic trauma and aortitis, or complicating a deep penetrating aortic ulcer, intraparietal hematoma, aortic aneurysm, and even aortic graft, often with atherosclerosis as a common background. METHODS: Given the relative rarity of this disease, an "inductive" retrospective method made it possible to retrieve clinical, radiological, and histopathological elements, which were mutually compared and validated through a "deductive" process of reinterpretation. RESULTS: We have identified three main structural constituents of this disease: a cavity, a single blood entry port, communicating with the aortic lumen, and a pseudocapsule. It is often caused by a chronic degenerative pathology of the intima and medial layers of the aorta, typically involving elastic fibers and smooth muscle cells, with possible intermediate stages of deep aortic ulcer or intraparietal hematoma. Otherwise, the acute onset may be secondary to acute aortitis or aortic injury. CONCLUSIONS: Today, thanks to the current angiographic tools represented by 3-D high resolution multidetector CT and MRI angiography, the diagnosis of thoracic aortic pseudoaneurysm is easier, as well as its surgical indications.
ABSTRACT
The human paraoxonase 2 (PON2) is the oldest member of a small family of arylesterase and lactonase enzymes, representing the first line of defense against bacterial infections and having a major role in ROS-associated diseases such as cancer, cardiovascular diseases, neurodegeneration, and diabetes. Specific Post-Translational Modifications (PTMs) clustering nearby two residues corresponding to pon2 polymorphic sites and their impact on the catalytic activity are not yet fully understood. Thus, the goal of the present study was to develop an improved PON2 purification protocol to obtain a higher amount of protein suitable for in-depth biochemical studies and biotechnological applications. To this end, we also tested several compounds to stabilize the active monomeric form of the enzyme. Storing the enzyme at 4 °C with 30 mM Threalose had the best impact on the activity, which was preserved for at least 30 days. The catalytic parameters against the substrate 3-Oxo-dodecanoyl-Homoserine Lactone (3oxoC12-HSL) and the enzyme ability to interfere with the biofilm formation of Pseudomonas aeruginosa (PAO1) were determined, showing that the obtained enzyme is well suited for downstream applications. Finally, we used the purified rPON2 to detect, by the direct molecular fishing (DMF) method, new putative PON2 interactors from soluble extracts of HeLa cells.
Subject(s)
Aryldialkylphosphatase , Proteomics , Aryldialkylphosphatase/metabolism , Aryldialkylphosphatase/chemistry , Humans , Proteomics/methods , Protein Refolding , Pseudomonas aeruginosa/enzymology , Enzyme Stability , Biofilms , Protein Processing, Post-TranslationalABSTRACT
BACKGROUND: Automatic exposure control (AEC) plays a crucial role in mammography by determining the exposure conditions needed to achieve specific image quality based on the absorption characteristics of compressed breasts. This study aimed to characterize the behavior of AEC for digital mammography (DM), digital breast tomosynthesis (DBT), and low-energy (LE) and high-energy (HE) acquisitions used in contrast-enhanced mammography (CEM) for three mammography systems from two manufacturers. METHODS: Using phantoms simulating various breast thicknesses, 363 studies were acquired using all available AEC modes 165 DM, 132 DBT, and 66 LE-CEM and HE-CEM. AEC behaviors were compared across systems and modalities to assess the impact of different technical components and manufacturers' strategies on the resulting mean glandular doses (MGDs) and image quality metrics such as contrast-to-noise ratio (CNR). RESULTS: For all systems and modalities, AEC increased MGD for increasing phantom thicknesses and decreased CNR. The median MGD values (interquartile ranges) were 1.135 mGy (0.772-1.668) for DM, 1.257 mGy (0.971-1.863) for DBT, 1.280 mGy (0.937-1.878) for LE-CEM, and 0.630 mGy (0.397-0.713) for HE-CEM. Medians CNRs were 14.2 (7.8-20.2) for DM, 4.91 (2.58-7.20) for a single projection in DBT, 11.9 (8.0-18.2) for LE-CEM, and 5.2 (3.6-9.2) for HE-CEM. AECs showed high repeatability, with variations lower than 5% for all modes in DM, DBT, and CEM. CONCLUSIONS: The study revealed substantial differences in AEC behavior between systems, modalities, and AEC modes, influenced by technical components and manufacturers' strategies, with potential implications in radiation dose and image quality in clinical settings. RELEVANCE STATEMENT: The study emphasized the central role of automatic exposure control in DM, DBT, and CEM acquisitions and the great variability in dose and image quality among manufacturers and between modalities. Caution is needed when generalizing conclusions about differences across mammography modalities. KEY POINTS: ⢠AEC plays a crucial role in DM, DBT, and CEM. ⢠AEC determines the "optimal" exposure conditions needed to achieve specific image quality. ⢠The study revealed substantial differences in AEC behavior, influenced by differences in technical components and strategies.
Subject(s)
Mammography , Radiographic Image Enhancement , Radiation Dosage , Radiographic Image Enhancement/methods , Mammography/methods , Phantoms, ImagingABSTRACT
Pseudomonas aeruginosa is one of the six antimicrobial-resistant pathogens known as "ESKAPE" that represent a global threat to human health and are considered priority targets for the development of novel antimicrobials and alternative therapeutics. The virulence of P. aeruginosa is regulated by a four-chemicals communication system termed quorum sensing (QS), and one main class of QS signals is termed acylhomoserine lactones (acyl-HSLs), which includes 3-Oxo-dodecanoil homoserine lactone (3-Oxo-C12-HSL), which regulates the expression of genes implicated in virulence and biofilm formation. Lactonases, like Paraoxonase 2 (PON2) from humans and the phosphotriesterase-like lactonases (PLLs) from thermostable microorganisms, are able to hydrolyze acyl-HSLs. In this work, we explored in vitro and in an animal model the effect of some lactonases on the production of Pseudomonas virulence factors. This study presents a model of chronic infection in which bacteria were administered by feeding, and Drosophila adults were treated with enzymes and the antibiotic tobramycin, alone or in combination. In vitro, we observed significant effects of lactonases on biofilm formation as well as effects on bacterial motility and the expression of virulence factors. The treatment in vivo by feeding with the lactonase SacPox allowed us to significantly increase the biocidal effect of tobramycin in chronic infection.
Subject(s)
Pseudomonas Infections , Pseudomonas aeruginosa , Humans , Animals , Drosophila melanogaster/metabolism , Persistent Infection , Biofilms , Quorum Sensing , Virulence Factors/genetics , Lactones/pharmacology , Bacteria/metabolism , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Tobramycin/pharmacologyABSTRACT
The problem of biofilm formation is a serious concern under various pathological conditions such as extensive burns, wounds in diabetic patients, bedsores, cystic fibrosis, nosocomial infections from implantable medical devices such as catheters, valves, etc. Environmental diffusion of biofilm (in pools, wet floors, industrial food plants) that could represent a reservoir of antibiotic resistant bacteria constitues an additional issue. In this work is described a lactonase from Rhodococcus erythropolis, a phosphotriesterase-like lactonase (PLL) enzyme, which has already been studied in the past and can be used for containment of biofilm formation. The protein is 28% and 40% identical with respect to the Pseudomonas diminuta PTE and the thermostable Saccharolobus solfataricus SsoPox respectively. The protein was obtained starting from a synthetic His-tagged gene, expressed in E. coli, purified and further characterized. New properties, not previously known or deducible from its sequence, have been highlighted. These properties are: the enzyme is thermophilic and thermostable even though it originates from a mesophilic bacterium; the enzyme has a long (months) shelf life at 4 °C; the enzyme is not only stable to low concentrations of the oxidant H2O2 but even activated by it at high concentrations; the enzyme proved to be a proficient quorum quenching enzyme, able to hydrolase acyl-homoserine lactones 3oxoC12-HSL and C4-HSL, and can inhibit up to 60% the formation of Pseudomonas aeruginosa (PAO1) biofilm. These different properties make the lactonase useful to fight resistant bacteria that induce inflammatory and infectious processes mediated by the quorum sensing mechanism.
Subject(s)
Phosphoric Triester Hydrolases , Quorum Sensing , Humans , Phosphoric Triester Hydrolases/genetics , Phosphoric Triester Hydrolases/metabolism , Escherichia coli/metabolism , Hydrogen Peroxide , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/metabolism , Biofilms , Bacteria/metabolism , Enzyme StabilityABSTRACT
BACKGROUND: In recent decades, the use of pesticides in agriculture has increased at a fast pace, highlighting safety problems for the environment and human health, which in turn has made it necessary to develop new detection and decontamination systems for pesticides. METHODS: A new qualitative test capable of detecting the presence of pesticides on fruits and vegetables by using thermostable enzymes was discovered, and the test was carried out on apples and aubergines. The contaminating pesticides were extracted from fruits with acetonitrile and analyzed with a biosensor system based on the thermostable esterase EST2 immobilized on a nitrocellulose filter. This enzyme is irreversibly inhibited mainly in the presence of organophosphates pesticides. Therefore, by observing esterase activity inhibition, we revealed the presence of residual pesticides on the fruits and vegetables. RESULTS: By analyzing the rate of esterase activity inhibition, we predicted that residual pesticides are present on the surface of the fruits. When we cleaned the fruits by washing them in the presence of the phosphotriesterase SsoPox before the detection of the esterase activity on filters, we observed a full recovery of the activity for apples and 30% for aubergines, indicating that the enzymatic decontamination of organophosphates pesticides took place. CONCLUSIONS: The reported method permitted us to assess the pesticides present on the vegetables and their decontamination.
ABSTRACT
The utility of pesticides in the agricultural field is unquestionable, but at the same time pesticide use presents serious hazards to the environment and the human health. For that reason, detection of pesticides and their biotransformation products in food is of utmost importance. According to previous studies, esterase-based biosensors have been proposed as a viable and efficient solution for the detection of organophosphate pesticides. In this project, a double mutant of the thermostable esterase-2 (EST2) from Alicyclobacillus acidocaldarius was studied as a potential biosensor, for its ability to detect residual amounts of pesticides. Initial characterisation of the enzyme was performed, that included determination of optimal pH, thermophilicity, as well as kinetic analysis. Subsequently, the enzyme was studied by enzymatic activity assays with and without the presence of various organophosphate compounds. The effect of the organophosphates on the enzymatic activity was measured and complete inhibition of the enzyme was observed after incubation with paraoxon. These experiments were followed by an additional method involving labelling of the enzyme with a fluorescent probe. In this case, the effect of different pesticides on the EST2 enzyme was monitored by measuring the fluorescence quenching upon addition to the enzyme. Fourteen compounds were screened with this method and significant fluorescence quenching was observed in the presence of paraoxon and methyl-paraoxon when used in equimolar amounts with the enzyme in the range of nanomolar. This biosensor has been also used to test the presence of pesticides in real food samples, like fruits and juices. This research represents a starting point to develop effective fluorescence-based biosensors aiming at the screening of mutants with different pesticide selectivity profiles. The use of this enzyme-based biosensor can have applications in the field of food traceability as well as environmental monitoring, to control the presence of toxic chemicals, in particular organophosphate pesticides.
ABSTRACT
Cellular functions are regulated through the gene expression program by the transcription of new messenger RNAs (mRNAs), alternative RNA splicing, and protein synthesis. To this end, the post-translational modifications (PTMs) of proteins add another layer of complexity, creating a continuously fine-tuned regulatory network. ADP-ribosylation (ADPr) is an ancient reversible modification of cellular macromolecules, regulating a multitude of key functional processes as diverse as DNA damage repair (DDR), transcriptional regulation, intracellular transport, immune and stress responses, and cell survival. Additionally, due to the emerging role of ADP-ribosylation in pathological processes, ADP-ribosyltransferases (ARTs), the enzymes involved in ADPr, are attracting growing interest as new drug targets. In this review, an overview of human ARTs and their related biological functions is provided, mainly focusing on the regulation of ADP-ribosyltransferase Diphtheria toxin-like enzymes (ARTD)-dependent RNA functions. Finally, in order to unravel novel gene functional relationships, we propose the analysis of an inventory of human gene clusters, including ARTDs, which share conserved sequences at 3' untranslated regions (UTRs).
Subject(s)
ADP-Ribosylation , RNA , ADP Ribose Transferases/genetics , Biology , Humans , Protein Processing, Post-Translational , RNA/metabolismABSTRACT
The development of faster, sensitive and real-time methods for detecting organophosphate (OP) pesticides is of utmost priority in the in situ monitoring of these widespread compounds. Research on enzyme-based biosensors is increasing, and a promising candidate as a bioreceptor is the thermostable enzyme esterase-2 from Alicyclobacillus acidocaldarius (EST2), with a lipase-like Ser-His-Asp catalytic triad with a high affinity for OPs. This study aimed to evaluate the applicability of Förster resonance energy transfer (FRET) as a sensitive and reliable method to quantify OPs at environmentally relevant concentrations. For this purpose, the previously developed IAEDANS-labelled EST2-S35C mutant was used, in which tryptophan and IAEDANS fluorophores are the donor and the acceptor, respectively. Fluorometric measurements showed linearity with increased EST2-S35C concentrations. No significant interference was observed in the FRET measurements due to changes in the pH of the medium or the addition of other organic components (glucose, ascorbic acid or yeast extract). Fluorescence quenching due to the presence of paraoxon was observed at concentrations as low as 2 nM, which are considered harmful for the ecosystem. These results pave the way for further experiments encompassing more complex matrices.
Subject(s)
Biosensing Techniques , Insecticides , Pesticides , Ecosystem , Fluorescence Resonance Energy Transfer , Paraoxon/toxicity , Pesticides/analysisABSTRACT
The widespread use of pesticides in the last decades and their accumulation into the environment gave rise to major environmental and human health concerns. To address this topic, the scientific community pointed out the need to develop methodologies to detect and measure the presence of pesticides in different matrices. Biosensors have been recently explored as fast, easy, and sensitive methods for direct organophosphate pesticides monitoring. Thus, the present work aimed at designing and testing a 3D printed adapter useful on different equipment, and a membrane support to immobilize the esterase-2 from Alicyclobacillus acidocaldarius (EST2) bioreceptor. The latter is labelled with the IAEDANS, a bright fluorescent probe. EST2 was selected since it shows a high specificity toward paraoxon. Our results showed good stability and replicability, with an increasing linear fluorescent intensity recorded from 15 to 150 pmol of labelled EST2. Linearity of data was also observed when using the immobilized labelled EST2 to detect increasing amounts of paraoxon, with a limit of detection (LOD) of 0.09 pmol. This LOD value reveals the high sensitivity of our membrane support when mounted on the 3D adapter, comparable to modern methods using robotic workstations. Notably, the use of an independent support significantly simplified the manipulation of the membrane during experimental procedures and enabled it to match the specificities of different systems. In sum, this work emphasizes the advantages of using 3D printed accessories adapted to respond to the newest research needs.
Subject(s)
Enzymes, Immobilized/metabolism , Esterases/metabolism , Organophosphorus Compounds/analysis , Pesticides/analysis , Printing, Three-Dimensional , FluorescenceABSTRACT
Surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry is a variant of the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. It is used in many cases especially for the analysis of protein profiling and for preliminary screening of biomarkers in complex samples. Unfortunately, these analyses are time consuming and protein identification is generally strictly limited. SELDI-TOF analysis of mass spectra (SELYMATRA) is a web application (WA) developed to reduce these limitations by (i) automating the identification processes and (ii) introducing the possibility to predict proteins in complex mixtures from cells and tissues. The WA architectural pattern is the model-view-controller, commonly used in software development. The WA compares the mass value between two mass spectra (sample vs. control) to extract differences, and, according to the set parameters, it queries a local database to predict most likely proteins based on their masses and different expression amplification. The WA was validated in a cellular model overexpressing a tagged NURR1 receptor, being able to recognize the tagged protein in the profiling of transformed cells. A help page, including a description of parameters for WA use, is available on the website.
Subject(s)
Protein Array Analysis , Proteins , Protein Array Analysis/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Proteins/analysis , Biomarkers/analysis , SoftwareABSTRACT
The DING proteins are ubiquitous in the three domains of life, from mesophiles to thermo- and hyperthermophiles. They belong to a family of more than sixty members and have a characteristic N-terminus, DINGGG, which is considered a "signature" of these proteins. Structurally, they share a highly conserved phosphate binding site, and a three dimensional organization resembling the "Venus Flytrap", both reminding the ones of PstS proteins. They have unusually high sequence conservation, even between distantly related species. Nevertheless despite that the genomes of most of these species have been sequenced, the DING gene has not been reported for all the relative characterized DING proteins. Identity of known DING proteins has been confirmed immunologically and, in some cases, by N-terminal sequence analysis. Only a few of the DING proteins have been purified and biochemically characterized. DING proteins are heterogeneous for their wide range of biological activities and some show different activities not always correlated with each other. Most of them have been originally identified for different biological properties, or rather for binding to phosphate and also to other ligands. Their involvement in pathologies is described. This review is an update of the most recent findings on old and new DING proteins.
Subject(s)
Extremophiles/metabolism , Phosphate-Binding Proteins/metabolism , Amino Acid Sequence , Archaea/metabolism , Eukaryota/metabolism , Phosphate-Binding Proteins/chemistry , Phosphate-Binding Proteins/geneticsABSTRACT
PON1, PON2, and PON3 belong to a family of lactone hydrolyzing enzymes endowed with various substrate specificities. Among PONs, PON2 shows the highest hydrolytic activity toward many acyl-homoserine lactones (acyl-HL) involved in bacterial quorum-sensing signaling. Accordingly, defense against pathogens, such as Brevundimonas aeruginosa (B. aeruginosa), was postulated to be the principal function of PON2. However, recent findings have highlighted the importance of PON2 in oxidative stress control, inhibition of apoptosis, and the progression of various types of malignancies. This review focuses on all of these aspects of PON2.
ABSTRACT
We identified two unstable variants in the third exon of α-globin genes: Hb Bernalda/Groene Hart (HBA1:c.358C>T), and Hb Caserta (HBA2:c.79G>A) in cis to Hb Sun Prairie (HBA2:c.391G>C), also named Hb Southern Italy. These mutations occurred in the H helix of the α-globin that is involved in heme contacting, specific recognition of α-hemoglobin-stabilizing protein (AHSP), and α1ß1 interactions. The carriers showed α-thalassemia phenotype, but one also jaundice and cholelithiasis. Molecular identification of clusters of families in Southern Italy encouraged molecular characterization of mRNA, globin chain analyses, molecular modeling studies, and comparison with globin variants to understand the mechanisms causing the α-thalassemia phenotype. A normal amount of Hb Bernalda/Groene Hart mRNA were found, and molecular modeling highlighted additional H bonds with AHSP. For Hb Southern Italy, showing an unexpected α/ß biosynthetic ratio typical of the ß-thalassemia type, two different molecular mechanisms were shown: Reduction of the variant mRNA, likely due to the No-Go Decay for the presence of unused triplet ACG at cod 26, and protein instability due to the impairment of AHSP interaction. The UDP glucuronosyltransferase 1A (UGT1A1) genotyping was conclusive in the case of jaundice and cholelithiasis. Multiple approaches are needed to properly identify the mechanisms leading to unstable variants and the effect of a mutation.
Subject(s)
Hemoglobin A/genetics , Hemoglobins, Abnormal/genetics , Mutation , Thalassemia/genetics , Adolescent , Adult , Aged , Binding Sites , Blood Proteins/metabolism , Cells, Cultured , Child , Female , Glucuronosyltransferase/genetics , Hemoglobin A/chemistry , Hemoglobin A/metabolism , Hemoglobins, Abnormal/chemistry , Hemoglobins, Abnormal/metabolism , Humans , Italy , Male , Middle Aged , Molecular Chaperones/metabolism , Phenotype , Protein Binding , Protein Stability , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Thalassemia/pathologyABSTRACT
The activity of human paraoxonase 2 (PON2) is rapidly reduced in cells incubated with the bacterial quorormone 3-Oxo-dodecanoyl Homoserine Lactone (3OC12HSL), an observation that led to hypothesize a fast PON2 post-translational modification (PTM). Recently, we detected a 3OC12HSL-induced PTM in a cell-free system in which a crude extract from 3OC12HSL-treated HeLa cells was able to inactivate and ubiquitinate at position 144 a recombinant PON2. Here we show the occurrence of this and new PTMs on PON2 in HeLa cells. PTMs were found to gather nearby the two SNPs, A148G, and S311C, that are related to type-2 diabetes and its complications. Furthermore, we detected a PTM nearby a 12 amino acids region that is deleted in PON2 Isoform 2. An in vitro mutation analysis showed that the SNPs and the deletion are involved in PON2 activity and suggested a role of PTMs on its modulation, while a SAXS analysis pointed to Isoform 2 as being largely unstructured, compared to the wild type. Besides, we discovered a control of PON2 expression via a putative mRNA operon involving the Wilms tumor 1 associated protein (WTAP) and the E3 ubiquitin ligase (E3UbL) baculoviral IAP repeat-containing 3 (BIRC3).
Subject(s)
Aryldialkylphosphatase/genetics , Baculoviral IAP Repeat-Containing 3 Protein/metabolism , Cell Cycle Proteins/metabolism , Gene Expression Regulation , RNA Splicing Factors/metabolism , Transcription, Genetic , A549 Cells , Adenosine Diphosphate Ribose/metabolism , Amino Acid Sequence , Aryldialkylphosphatase/chemistry , Aryldialkylphosphatase/metabolism , Gene Silencing , HeLa Cells , Humans , Kinetics , Models, Biological , Models, Molecular , Operon/genetics , Peptides/chemistry , Peptides/metabolism , Polymorphism, Single Nucleotide/genetics , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Processing, Post-Translational , RNA, Messenger/genetics , RNA, Messenger/metabolism , Scattering, Small Angle , Ubiquitination , X-Ray DiffractionABSTRACT
Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from Saccharolobus solfataricus. The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 × 105 M-1 s-1 against paraoxon hydrolysis at 70°C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 Å. We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.
Subject(s)
Aryldialkylphosphatase/chemistry , Aryldialkylphosphatase/metabolism , Mutant Proteins/metabolism , Protein Multimerization , Sulfolobus solfataricus/enzymology , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/metabolism , Catalytic Domain , Circular Dichroism , Directed Molecular Evolution , Enzyme Stability , Hydrogen-Ion Concentration , Ions , Metals/chemistry , Models, Molecular , Nerve Agents/chemistry , Phosphoric Triester Hydrolases/chemistry , Phosphoric Triester Hydrolases/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Structural Homology, Protein , Structure-Activity Relationship , TemperatureABSTRACT
Pesticides represent some of the most common man-made chemicals in the world. Despite their unquestionable utility in the agricultural field and in the prevention of pest infestation in public areas of cities, pesticides and their biotransformation products are toxic to the environment and hazardous to human health. Esterase-based biosensors represent a viable alternative to the expensive and time-consuming systems currently used for their detection. In this work, we used the esterase-2 from Alicyclobacillus acidocaldarius as bioreceptor for a biosensing device based on an automated robotic approach. Coupling the robotic system with a fluorescence inhibition assay, in only 30 s of enzymatic assay, we accomplished the detection limit of 10 pmol for 11 chemically oxidized thio-organophosphates in solution. In addition, we observed differences in the shape of the inhibition curves determined measuring the decrease of esterase-2 residual activity over time. These differences could be used for the characterization and identification of thio-organophosphate pesticides, leading to a pseudo fingerprinting for each of these compounds. This research represents a starting point to develop technologies for automated screening of toxic compounds in samples from industrial sectors, such as the food industry, and for environmental monitoring.
Subject(s)
Biosensing Techniques/methods , Organophosphates/chemistry , Organophosphorus Compounds/chemistry , Robotics/methods , Alicyclobacillus/chemistry , Biological Assay/methods , Environmental Monitoring/methods , Esterases/chemistry , Fluorescence , Limit of Detection , Pesticides/chemistryABSTRACT
Organophosphate (OP) pesticides are widely used in the agricultural field and in the prevention of pest infestation in private and public areas of cities. Despite their unquestionable utility, several of these compounds demonstrate toxic effects to the environment and human health. In particular, the occurrence of some organophosphate pesticides is correlated to the incidence of nervous system disorders, especially in children. The detection of pesticide residues in the human body represents an important task to preserve human health. In our work we propose the use of esterase-based biosensors as a viable alternative to the expensive and time-consuming systems currently used for their detection in human fluids. Using the esterase-2 activity, coupled with a fluorescence inhibition assay, we are able to detect very low concentration levels of diethyl (4-nitrophenyl) phosphate (paraoxon) in the range of the femtomole (fmol). Method robustness tests indicate the stability of esterase-2 in a diluted solution of 4% human urine, and we are able to accurately determine concentration levels of paraoxon in the range from 0.1 to 2 picomoles (pmol). The system sensitivity for OP detection is calculated at 524 ± 14.15 fmol of paraoxon recognized at 10% of inhibition, with an estimated limit of quantification of 262 ± 8.12 pmol mL-1. These values are comparable with the most recent analysis methods based on mass spectrometry carried out on human samples for pesticide detection. This research represents a starting point to develop cheap and fast testing methods for a rapid screening of toxic substances in human samples.
Subject(s)
Paraoxon/urine , Biosensing Techniques/methods , Enzyme Assays/methods , Fluorescence , Humans , Insecticides/urine , Nitrophenols/urine , Organophosphates/urine , Organophosphorus Compounds/urine , Pesticides/urineABSTRACT
Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.
Subject(s)
Biosensing Techniques/methods , Chemical Warfare Agents/analysis , Decontamination/methods , Nerve Agents/analysis , Organophosphorus Compounds/analysis , Organophosphorus Compounds/metabolism , Pesticides/analysis , Alicyclobacillus/enzymology , Alicyclobacillus/genetics , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/metabolism , Chemical Warfare Agents/metabolism , Limit of Detection , Nerve Agents/metabolism , Pesticides/metabolism , Phosphoric Triester Hydrolases/genetics , Phosphoric Triester Hydrolases/metabolism , Sulfolobus acidocaldarius/enzymology , Sulfolobus acidocaldarius/genetics , Sulfolobus solfataricus/enzymology , Sulfolobus solfataricus/geneticsABSTRACT
The need to find alternative bioremediation solutions for organophosphate degradation pushed the research to develop technologies based on organophosphate degrading enzymes, such as phosphotriesterase. The use of free phosphotriesterase poses limits in terms of enzyme reuse, stability, and process development. The heterogenization of enzyme on a support and their use in bioreactors implemented by membranes seems a suitable strategy, thanks to the ability of membranes to compartmentalize, to govern mass transfer, and to provide a microenvironment with tuned physicochemical and structural properties. Usually, hydrophilic membranes are used since they easily guarantee the presence of water molecules needed for the enzyme catalytic activity. However, hydrophobic materials exhibit a larger shelf life and are preferred for the construction of filters and masks. Therefore, in this work, hydrophobic polyvinylidene fluoride (PVDF) porous membranes were used to develop biocatalytic membrane reactors (BMR). The phosphotriesterase-like lactonase (PLL) enzyme ( SsoPox triple mutant from S. solfataricus) endowed with thermostable phosphotriesterase activity was used as model biocatalyst. The enzyme was covalently bound directly to the PVDF hydrophobic membrane or it was bound to magnetic nanoparticles and then positioned on the hydrophobic membrane surface by means of an external magnetic field. Investigation of kinetic properties of the two BMRs and the influence of immobilized enzyme amount revealed that the performance of the BMR was mostly dependent on the amount of enzyme and its distribution on the immobilization support. Magnetic nanocomposite mediated immobilization showed a much better performance, with an observed specific activity higher than 90% compared to grafting of the enzyme on the membrane. Even though the present work focused on phosphotriesterase, it can be easily translated to other classes of enzymes and related applications.